7 - heteromonocyclic-substituted acylamido derivatives of desacetyl cephalosporanic acid



U.S. Cl. 260-243 2 Claims ABSTRACT OF THE DISCLOSURE Derivatives of 7heteromonocyclic substituted acylamido desacetylcephalosporanic acid aredisclosed. These compounds are useful antibacterial agents.

This application is a continuation-in-part of my applications Serial No.115,612, filed June 8, 1961, now abandoned and Serial No. 220,855 filedAug. 31, 1962, now U.S. Patent 3,218,318 issued Nov. 16, 1965.

This invention relates to novel organic compounds and to methods fortheir preparation.

The novel compounds of the present invention are 7-(heteromonocyclic-substituted acylamido) derivatives ofdesacetylcephalosporanic acid, having the following formula:

and the salts thereof with pharmaceutically acceptable cations, inwhich:

R is a monocyclic ring having at least one hetero atom of the classconsisting of 'O, S, and N; and n is zero or 1.

Thus, R can be dioxanyl, 2-furyl, 3-furyl, imidazolyl, isoxazolyl,morpholinyl, oxazolyl, pyranyl, pyrazinyl, pyrazolyl, N-pyridylZ-pyridyl, 3-pyridyl, pyrimidyl, N- pyrryl, Z-pyrryl, 3-pyrryl,thiazolyl, Z-thienyl, 3-thienyl, triazinyl, triazolyl and the like; thepartially and completely hydrogenated derivatives thereof such astetrahydrofuryl imidazolinyl, imidazolidyl, piperidyl,tetrahydropyrimidyl, pyrrolidyl, and the like; and the derivativesthereof having chlorine, fluorine, bromine, iodine nitro, methoxy,methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, or other C -Calkyl substituent at one or more positions on the ring, e.g., thepicolyls, the methylfuryls, the methylthienyls, nitrofuryl,nitropyridyl, nitrothienyl, chlorofuryl, bromopyridyl, fluoropyrryl,methoxypyrimidyl, and the like.

The novel compounds of the present invention are related tocephalosporin C insofar as they contain the 2,3- dihydro-6H-1,5-thiazinering with a fused B-lactam ring in the 5,6 position, which ischaracteristic of cephalosporin C. However, unlike cephalosporin C,which contains the -amino-N'-adipamy1 group in the 7 position, thecompounds of the present invention are characterized by aheteromonocylic-substituted acylamido group in the 7 position. Moreover,unlike cephalosporin C, which contains acetoxyrnethyl in the 3 position,the compounds of the present invention contain hydroxymethyl in the 3position, and may thus be termed derivatives of desacetylcephalosporanicacid," a term sometimes shortened to cephalosporadesic acid.

3,459,746 Patented Aug. 5, 1969 The compounds of the present inventionare highly effective antibacterial agents. They are characterized bypenicillinase-resistance, acid stability, and activity against a broadrange of microorganisms, including both grampositive and gram-negativepathogens, and are considerably less subject to serum binding than arethe analogous 3-acetoxymethyl compounds. They are conveniently preparedand administered in the form of the salts of the carboxyl group withpharmaceutically acceptable cations, including, for example,water-soluble salts such as the sodium, potassium, lithium, ammonium,and substituted ammonium salts, as well as the less water-soluble saltssuch as the calcium, barium, procaine, quinine, anddibenzylethylenediamine salts. Administration is preferably byintramuscular injection in isotonic saline at a dose (for adults) around0.25 to 0.50 g. every four to six hours.

The following examples will illustrate the types of compounds availablein accordance with the present invention:

7-fi-thienylacetamidocephalosporadesic acid7-;8-thienylcarboxamidocephalosporadesic acid7-a-furylacetamidocephalosporadesic acid7-a-furylcarboxamidocephalosporadesic acid 7- (2'-pyranylacetamid0cephalosporadesic acid 7- (2'-morpholinylacetamido) cephalosporadesicacid 7-(3-bromo-2'-pyridylacetamid0) cephalosporadesic acid 7-5-methoxy-3'-pyridylacetarnido cephalosporadesic acid7-uthienylcarboxamidocephalosporadesic acid7-(2-pyrazinylacetamido)cephalosporadesic acid 7-(2-pyridylacetamido)cephalosporadesic acid 7- (or-picolyl-3 '-acetamido cephalosporadesicacid 7-fl-furylcarboxamidocephalosporadesic acid 7-(2'-triazinylacetamido cephalosporadesic acid 7- (4 5-imidazolylacetamido cephalo sporadesic acid 7-(2'-piperidylacetamido)cephalosporadesic acid7-fl-(5-pyrimidyl)acetamidocephalosporadesic acid7-fi-furylacetamidocephalosporadesic acid 7- (2'-methyl-3-furylacetamido cephalosporadesic acid 7- 5 -oxazolylacetamidocephalosporadesic acid 7- 3'methyl-2'-thienylacetamido)cephalosporadesic acid 7- 5 -nitro-2'-thienylacetamido cephalosporadesicacid 7-,8-(5'-fluoro-2'-pyridy1)acetamidocephalosporadesic acid 7-5'-imidazolinylacetamido) cephalosporadesic acid7-(2-thiazolylacetamido)cephalosporadesic acid and the like.

The source material for the compounds of the present invention iscephalosporin C, more precisely known as 7-(5'-amino-adipamido)cephalosporanic acid, which can be prepared bycultivating a cephalosporin C-producing organism in a suitable nutrientmedium, as described in British patent specification 810,196, publishedMar. 11, 1959.

Cephalosporin C is readily converted into the corresponding nucleuscompound, 7-aminocephalosporanic acid, by cleaving the5'-amino-N-adipamyl side chain between its amido carbonyl group and itsamido nitrogen, suitably by reacting cephalosporin C with nitrosylchloride in formic acid, then hydrolytically cleaving, according to themethod of Morin et al. described in U.S. Patent 3,188,311 (June 8,1965).

The nucleus thus obtained is conveniently converted into a7-acylamidocephalosporanic acid as desired by acylation. For thispurpose, any of the conventional acylation procedures can be employed,utilizing any of the various types of known acylating agents having thecomposition WhlCh yields the desired side chain. A convenient acylatingagent is the appropriate acyl chloride or bromide. The acylation iscarried out in water or an appropriate organic solvent, preferably undersubstantially neutral conditions, and preferably at reduced temperature,i.e.,

above the freezing point of the reaction mixture and up to about 20 C.In a typical procedure, 7-aminocephalosporanic acid is dissolved inwater with a sufficient quantity of sodium bicarbonate or otherappropriate alkali to promote solution, the concentration of the7-aminocephalosporanic acid being about 1 to about 4 percent by weight.The solution is cooled to around to 5 C., and a solution of theacylating agent is added in about 20 percent excess, with stirring andcooling. The pH of the mixture can be maintained, if it tends to vary,around the neutral level by bubbling carbon dioxide therein. Afteraddition of the acylating agent has been completed, stirring of thereaction mixture is continued and the mixture is allowed to warm to roomtemperature. The reaction product is then acidified to around pH 2 andextracted with an organic solvent such as ethyl acetate. The ethylacetate extract is back-extracted with water at pH 5.5 to 6, employingfor pH adjustment a base containing the cation of the desired finalproduct. The water solution is separated and evaporated substantially todryness. The residue is taken up in a minimum quantity of water and theacylation product is precipitated by adding a large excess of acetone,and if necessary ether. The crystalline product obtained thereby isfiltered, washed with acetone, and dried.

The acylation can also be carried out with the corresponding carboxylicacid, employed in conjunction with an equimolar proportion of acarbodiimide such as N,N'- diisopropylcarbodiimide, N,N'dicyclohexylcarbodiimide, N,N' bis (p dimethylaminophenyl)carbodiimide,N-ethyl-N-(4"-ethylmorpholinyl)carbodiimide, or the like, and theacylation proceeds at ordinary temperatures in such cases.Alternatively, the carboxylic acid can be converted into thecorresponding acid anhydride, or into the azide, or into an activatedester, and any of these derivatives can be used to etfect the desiredacylation. Other agents can readily be ascertained from the art.

Many of the acylating agents, together with methods for theirpreparation, are described in the literature, and a number of them arecommercially available. All of them are readily prepared by methods wellknown in the art.

In many cases, the acylating agent may contain one or more asymmetriccarbon atoms and thus exist in optically active forms. When prepared byordinary chemical means, such compounds are ordinarily obtained inracemic form i.e., an equimolar mixture of the optical isomers, havingno optical rotation. When the separate optical isomers are desired, theacylating agent can be resolved in a conventional manner such as byreacting the free acid with cinchonine, strychnine, brucine, or thelike, then fractionally crystallizing to separate the diasteroisomericsalts, and separately acidifying the solid phase and the liquid phase toliberate the optical isomers. The free acids thus obtained can beemployed as such for the acylation, preferably in conjunction with acarbodiimide, or may be converted by conventional means into thecorresponding acid halide or into a mixed anhydride, care beingexercised to avoid extremes of conditions which might produceracemization.

The 7-acylamidocephalosporanic acids obtained in the foregoing mannerare conveniently converted into the desired 7-acylamidocephalosporadesicacids by treatment with citrus acetylesterase for several hours inaqueous phosphate buffer at pH 6.5-7 according to the method of Jansen,Jang, and MacDonnell, Archiv. Biochem., 15 (1947), 415-31.

The invention will be more readily understood from the followingoperating example, which is submitted only as an illustration, and notby way of limitation.

Example 7-aminocephalosporanic acid (12 g.) was suspended in a mixtureof 200 ml. of water and 160 ml. of acetone and cooled in an ice bath. Tothe suspension were added 7.5 g. of sodium bicarbonate with stirring, asa result of which the solids went into solution. To the cooled solutionwere 4 added 6.5 g. of ot-thienylacetyl chloride dissolved in 40 ml. ofacetone over a period of one hour, after which the reaction mixture wasstirred in the cold for two additional hours. The reaction productmixture was then stripped of acetone under vacuum, and 200 ml. of ethylacetate were added, followed by 1 N hydrochloric acid to pH 2. Themixture was filtered and the aqueous phase was separated and discarded.The ethyl acetate phase was stirred with 100 ml. of water and adjustedto pH 6.5 with aqueous 1 N potassium hydroxide solution, 45 ml. beingrequired. The resulting aqueous extract was separated and evaporated todryness under vacuum. The residue was recrystallized from a mixture ofmethanol and isopropyl alcohol. The resulting intermediate,7-a-thienylacetamidocephalosporanic acid potassium salt, weighed 9.1 g.

An orange flavedo solution which had been prepared by the method ofJansen, Jang, and MacDonnell, referred to above, was further purified inthe following way. To the solution (approximately 340 ml.) were added17.5 g. of Darco G60 activated carbon, and the mixture was stirredslowly for 15 minutes in the cold, then centrifuged in the cold at15,000 rpm. The supernatant liquid (285 ml.) was poured off, and to itwere slowly added 49.9 g. of ammonium sulfate crystals with stirring,giving a solution 30 percent saturated with ammonium sulfate. Themixture was again centrifuged and the solid was discarded. To thesupernatant liquid (305 ml.) were slowly added 60 g. of ammonium sulfatewith cooling and stirring, giving a solution 60 percent saturated withammonium sulfate. The mixture was again centrifuged, and the supernatantliquid was discarded. The solids were dissolved in cold water andbrought to a volume of ml. The result was a stable solution of purifiedcitrus acetylesterase.

7 u-thienylacetamidocephalosporanic acid potassium salt (2.0 g.) wasdissolved in water, adjusted to pH 7 with aqueous 1 N sodium hydroxidesolution, and diluted to 86 ml. with water. The resulting solution was0.05 molar in the dissolved compound. The solution was commingled with21.5 ml. of 0.1 M phosphate buffer at pH 7, then with 43 ml. of citrusacetylesterase which had been prepared and purified as described aboveand adjusted to pH 7. The solution volume was adjusted to 430 ml. withwater, then held in a beaker at 37 C. with slow stirring. The pH wasmaintained at 7 by dropwise addition of aqueous 1 N sodium hydroxidesolution. Exposure to these conditions was continued for a period of 4hours, this being one hour past the time at which a parallel Warburgcontrol (described below) indicated completion of the reaction.

The Warburg control was run in the following manner. Into the body of aWarburg flask were placed 0.4 ml. of the original solution of 7 ozthienylacetamidocephalosporanic acid potassium salt and 1.3 ml. ofWater. Into the side arm was placed 0.2 ml. of the citrus acetylesterasepreparation. An aqueous 1 M solution of sodium bicarbonate was saturatedwith carbon dioxide, and 0.25 ml. was added to the body of the flask,and 0.05 ml. to the side arm. The manometer, having been purged withcarbon dioxide, was immediately attached. The gas was allowed to flowfor 15 minutes at room tempearture, and was then turned off. Theapparatus was allowed to equilibrate 15 minutes in the 37 C.constant-temperature bath, the side arm being open at first to permitventing, then closed. The contents of the side arm were then poured intothe body of the flask and rinsed back and forth, thus initiating thedesacetylation reaction, as evidenced by the evolution of carbondioxide. Gas evolution ceased at the end of 3 hours, and it wasdetermined that a total of 377 ml. (corrected) of carbon dioxide hadbeen evolved, corresponding to 16.8 millimoles or 84 percent of theory.

The product solution (approximately 400 ml.) was salted with sodiumchloride (40 g.), cooled, overlayered with an equal volume of ethylacetate, and acidified to pH 2.0 with 1 N hydrochloric acid. The mixturewas filtered to break the suspension and the layers were separated. The

organic phase was adjusted in the cold to pH 5.8 with aqueous 1 Npotassium hydroxide solution and the layers were separated. The aqueousextract was evaporated to dryness at reduced pressure and mildlyelevated temperature. The residue was dissolved in hot methanol,filtered, diluted with isopropyl alcohol, and concentrated. Theresulting white solid was filtered oil and dried. Yield, 0.95 g. Thefiltrate was further concentrated and chilled, and a second crop wasobtained weighing 200 mg.

'lhe product, 7 a-thienylacetamidocephalosporadesic acid potassium salt,was found to be effective against penicillin-resistant Staphylococcusaureus, having the following minimum inhibitory concentrations, both inthe presence and in the absence of human blood serum, against fourclinical isolates, as measured by the gradient-plate technique andexpressed in micrograms per milliliter:

Strain V-30 V-32 V-41 V-84 Without serum 0. 5 4 0. 5 2 With serum 2 2 23 The product was also shown to be effective in varying degrees againsta variety of other organisms.

Minimum inhibitory Minimum inhibitory Organism: concentration, meg/ml.Shigella sonnei 62 Escherichia coli N-lO 134 E. coli N-26 89 I claim:

1. An antibiotic substance having the following formula:

wherein R is a monocyclic ring having at least one hetero atom of theclass consisting of O, S, and N selected from the group consisting ofB-thienyl, a-thienyl, a-furyl, 2- pyranyl, 2'-morpholinyl,3'-bromo-2'-pyridyl, 5-methoxy- 3-pyridyl, 2-pyrazinyl, 2'-pyridyl,a-picolyl, p-furyl, 2'- triazinyl, 4-(5')-imidazolyl, 2-piperidyl,5'-pyrimidyl, 2'- methyl-3'-furyl, 5-oxazolyl, 3-methyl-2-thienyl,5-nitro- 2'-pyridyl, 5-fluoro-2-pyridyl, 5-imidazolinyl, and 2-thiazolyl.

2. 7-a-thienylacetamidocephalosporadesic acid.

References Cited UNITED STATES PATENTS 3,196,151 7/1965 Hoover et a1.3,218,318 11/1965 Flynn.

NICHOLAS S. RIZZO, Primary Examiner US. Cl. X.R. 260-999

